One of the primary goals of the fields of synthetic biology and metabolic engineering is to reprogram the behavior of organisms via the insertion of rationally engineered synthetic gene networks or the alteration of naturally occurring metabolic networks. A prominent component of this process is the need to balance gene expression to obtain optimal yields of desired products and avoid buildup of toxic intermediates. This proposal will address the current lack of methodology to serve this need by developing techniques for analyzing differences in dynamic gene expression and methods for accounting for and correcting those differences in the context of a metabolic pathway. The transient signaling molecule nitric oxide (NO) will be used for this purpose because it can be produced by NO synthases and degraded by NO dioxygenases or reductases, resulting in a tunable signal that will serve as an indicator of the difference in gene expression between two promoters. Coupled with a NO-sensitive promoter that will be developed as part of this research, the presence of NO will be used to drive gene expression of additional enzymes in response to an imbalance in the levels of NO production and degradation. As a test of this proposed system, I will apply it towards the production of 1-butanol (a potential biofuel) from threonine via the keto-acid biosynthesis metabolic pathway. In addition to the applications of this proposed research to synthetic biology and metabolic engineering, the development of a NO sensor for use in vivo will have applications to studies of bacterial antibiotic resistance and mammalian cell signaling and the development of a system to balance gene expression may have applications in the study of gene therapy. PUBLIC HEALTH RELEVANCE: Dynamic balancing of gene expression in metabolic pathways and synthetic networks is important for the regulated production of desired products, such as biofuels and biologically produced pharmaceuticals. This proposed research will provide a novel tool for analyzing differences in gene expression and balancing expression levels in a variety of host organisms by use of the small signaling molecule nitric oxide as a sensitive and transient indicator of expression ratios. Successful completion of this project will provide a versatile nitric oxide sensor that can be used to regulate gene expression in response to nitric oxide in vivo, and an analysis system of dynamic gene expression for use in identifying and balancing expression differences in metabolic pathways.